Ligand-Independent Recruitment of SRC-1 to Estrogen Receptor β through Phosphorylation of Activation Function AF-1  André Tremblay, Gilles B Tremblay, Fernand Labrie, Vincent Giguère  Molecular Cell  Volume 3, Issue 4, Pages 513-519 (April 1999) DOI: 10.1016/S1097-2765(00)80479-7

Figure 1 Basal ERβ Transcriptional Activation by SRC-1 Is AF-2 Independent Pure antiestrogen EM-652 but not the partial antagonist OHT inhibits basal ERβ transcriptional activation by SRC-1. Cos-1 cells were transfected with ERETKLuc along with equivalent amounts of ERβ and SRC-1 expression plasmids and incubated with 100 nM OHT or EM-652. Basal activity of an ERβ AF-2 mutant is induced by SRC-1. Cos-1 cells were transfected with ERETKLuc reporter and equivalent amounts of ERβ or ERβ L509A AF-2 mutant and SRC-1 expression plasmids. Cells were then treated with 10 nM E2 or left untreated for 16 hr prior to harvest. Molecular Cell 1999 3, 513-519DOI: (10.1016/S1097-2765(00)80479-7)

Figure 2 The Ligand-Independent Effect of SRC-1 Resides in the A/B Region of ERβ and Is Transposable (A) Representation of the various constructs used. Numbering of mouse ERβ is relative to its longest form. (B) Cos-1 cells were transfected with expression plasmids encoding the various ERα and β derivatives and SRC-1, and transcriptional activities were measured on an ERETKLuc reporter. Cells were treated with 10 nM E2 or left untreated for 16 hr prior to harvest. Molecular Cell 1999 3, 513-519DOI: (10.1016/S1097-2765(00)80479-7)

Figure 3 Interaction of SRC-1 with ERβ AF-1 (A) Region 104–122 of ERβ AF-1 mediates ligand-independent activation by SRC-1. Various N- and C-terminal deleted forms of ERβ within the A/B region were tested in transfection on an ERETKLuc in Cos1 cells in the presence of SRC-1 expression vector. (B) SRC-1 associates with the A/B region of ERβ in vitro. Pull-down experiments were performed with in vitro translated and 35S-labeled SRC-1. The input lane (i) represents 5% of the labeled SRC-1 used in each experiment. (C) Identification of two consensus Pro-directed kinase sites at positions 106 and 124 in ERβ AF-1 of which the motif comprising Ser-106 is conserved with other steroid receptors within the A/B region. Numbering of human ERβ is relative to its longest form, and of PR to PR-B. (D) Ser-106 and Ser-124 are target sites for MAPK. GST fusions of wild-type and serine-mutated ERβ (aa 96–130) were in vitro phosphorylated by Erk2. MBP was added as a MAPK control substrate. Molecular Cell 1999 3, 513-519DOI: (10.1016/S1097-2765(00)80479-7)

Figure 4 MAPK-Mediated Phosphorylation of ERβ AF-1 Increases SRC-1 Recruitment In Vitro and In Vivo (A) Phosphorylation of ERβ AF-1 increases the binding to SRC-1 in vitro. Wild-type and mutated GST-ABCβ fusions bound to glutathione beads were incubated in the presence (+) or absence (−) of Erk2 and subjected to pull-down assay with in vitro translated and 35S-labeled SRC-1. Equal amounts of proteins were loaded except for the input (i), which represents 5% of proteins used. (B) Effects of kinase inhibitors and point mutations on the transcriptional activity of ERβ by SRC-1. Cos-1 cells were transfected with ERETKLuc reporter and ERβ (aa 65–549) or ERβ AF-1 mutants in the absence or presence of SRC-1 expression vector. In the case of kinase inhibitors, cells were treated either with 50 μM PD98059 (PD) or 1 μM staurosporin (ST) 20 min before harvest. (C) ERβ AF-1 recruits SRC-1 in vivo. 293 T cells were cotransfected with expression vectors encoding HA-tagged SRC-1 and AF-2-defective (L509A) ERβ mutants in the absence or presence of constitutively activated Ras (rasV12) expression vectors. Cells were fed with growth medium for 48 hr and harvested 30 min following treatment with 50 μM PD or vehicle alone. The immunoprecipitates were subjected to Western analysis using an anti-ERβ antibody. As a control, whole-cell extracts of each sample were analyzed by Western blotting to monitor the amounts of ERβ (α-ERβ) and SRC-1 (α-SRC-1) expressed in transfected cells. (D) EGF induces recruitment of SRC-1 by ERβ AF-1. 293 T cells were transfected with HA-tagged SRC-1 and AF-2-defective (L509A) ERβ mutant expression vectors. Thirty minutes prior to harvest, cells were treated with 20 ng/ml EGF, 50 μM PD, or left untreated. Cell lysates were then analyzed by immunoprecipitation and Western blotting as in Figure 4C. (E) In vivo phosphorylation of ERβ AF-1. Subconfluent 293 T cells were transfected with wild-type or mutated ABCDβ (aa 65–274) expression plasmids, and refed with DMEM. Forty-eight hours later, the transfected cells were metabolically labeled with [32P] orthophosphate and treated with EGF (20 and 50 ng/ml) or 50 μM PD, or left untreated. Cells were harvested and subjected to immunoprecipitation with an antibody to ERβ. Molecular Cell 1999 3, 513-519DOI: (10.1016/S1097-2765(00)80479-7)